Membrane-associated guanylate kinase-like properties of β-subunits required for modulation of voltage-dependent Ca ²⁺ channels

High-voltage-activated Ca2+ channels regulate diverse functions ranging from muscle contraction to synaptic transmission. Association between auxiliary beta- and distinct pore-forming alpha1-subunits is obligatory for forming functional high-voltage-activated Ca2+ channels, yet the structural determinants underlying this interaction remain poorly understood. Recently, homology modeling of Ca(2+)-channel beta1b-subunit identified src homology 3 (SH3) and guanylate kinase (GK) motifs in a tandem arrangement reminiscent of the membrane-associated guanylate kinase (MAGUK) class of scaffolding proteins. However, direct evidence for MAGUK-like properties and their functional implications in beta-subunits is lacking. Here, we show a functional requirement for both SH3 and GK domains in beta2a. Point mutations in either the putative beta2a SH3 or GK domains severely blunted modulation of recombinant L-type channels, showing the importance of both motifs for a functional alpha1-beta interaction. Coexpression of these functionally deficient beta2a-SH3 and GK mutants rescued WT currents, demonstrating trans complementation similar to that observed in MAGUKs. Truncated "hemi-beta2a" subunits, containing either the SH3 or GK domain, were ineffective on their own, but reconstituted WT currents when coexpressed. Moreover, the SH3 and GK domains were found to interact in vitro. These findings reveal MAGUK-like properties in beta-subunits that are critical for alpha1-subunit modulation, revise current models of alpha1-beta association, and predict new physiological dimensions of beta-subunit function.